Enhanced nutrient transport improves the depth-dependent properties of tri-layered engineered cartilage constructs with zonal co-culture of chondrocytes and MSCs

Abstract

Biomimetic design in cartilage tissue engineering is a challenge given the complexity of the native tissue. While numerous studies have generated constructs with near-native bulk properties, recapitulating the depth-dependent features of native tissue remains a challenge. Furthermore, limitations in nutrient transport and matrix accumulation in engineered constructs hinders maturation within the central core of large constructs. To overcome these limitations, we fabricated tri-layered constructs that recapitulate the depth-dependent cellular organization and functional properties of native tissue using zonally derived chondrocytes co-cultured with MSCs. We also introduced porous hollow fibers (HFs) and HFs/cotton threads to enhance nutrient transport. Our results showed that tri-layered constructs with depth-dependent organization and properties could be fabricated. The addition of HFs or HFs/threads improved matrix accumulation in the central core region. With HF/threads, the local modulus in the deep region of tri-layered constructs nearly matched that of native tissue, though the properties in the central regions remained lower. These constructs reproduced the zonal organization and depth-dependent properties of native tissue, and demonstrate that a layer-by-layer fabrication scheme holds promise for the biomimetic repair of focal cartilage defects.

Statement of significance: Articular cartilage is a highly organized tissue driven by zonal heterogeneity of cells, extracellular matrix proteins and fibril orientations, resulting in depth-dependent mechanical properties. Therefore, the recapitulation of the functional properties of native cartilage in a tissue engineered construct requires such a biomimetic design of the morphological organization, and this has remained a challenge in cartilage tissue engineering. This study demonstrates that a layer-by-layer fabrication scheme, including co-cultures of zone-specific articular CHs and MSCs, can reproduce the depth-dependent characteristics and mechanical properties of native cartilage while minimizing the need for large numbers of chondrocytes. In addition, introduction of a porous hollow fiber (combined with a cotton thread) enhanced nutrient transport and depth-dependent properties of the tri-layered construct. Such a tri-layered construct may provide critical advantages for focal cartilage repair. These constructs hold promise for restoring native tissue structure and function, and may be beneficial in terms of zone-to-zone integration with adjacent host tissue and providing more appropriate strain transfer after implantation.

Keywords: Cartilage tissue engineering; Co-culture; Depth-dependent properties; Hyaluronic acid; Mesenchymal stem cells; Nutrient transport; Zonal chondrocytes.

Figure. 1. A Schematic of a tri-layered construct fabrication with zonal CH/MSC co-culture

(A) Zonal chondrocytes (SZ, MZ and DZ; left) and MSCs (right) were isolated from articular cartilage and bone marrow, respectively. Isolated cells were expanded in culture separately and labeled with CellTracker (SZ: red, MZ: purple, DZ: green and MSC: blue) to trace the distribution of cell subpopulations during long-term culture. (B) Fabrication procedure for single- and tri-layered constructs.

Figure. 2. Development and maturation of a tri-layered construct

(A) A cross-sectional view of a tri-layered construct showing distinct zonal sub-layers visualized by CellTracker at 16 weeks (SZ CH: red, MZ: purple, DZ: green and MSC: blue; Top layer: SZ-MSC, Middle: MZ-MSC and Bottom: DZ-MSC; scale bar = 1 mm). (B–C) Zoomed views of the dashed boxes showing cells and interface of (B) top-middle and (C) middle-bottom layer (scale bar = 100 µm). Co-cultures of SZ-MSC (Inset B) and DZ-MSC (Inset C) were evident in each sub-layer (20×; scale bar = 100 µm). (D) Immunohistochemistry of type II collagen (top) and chondroitin sulfate (bottom) at 8 weeks (60 million cells/mL; CH:MSC = 1:4; scale bar = 1 mm). (E) Heterogeneous matrix accumulation at 8 weeks in a tri-layered construct due to limited nutrient transport to the central region. The peripheral region (solid arrow) showed denser matrix and less swelling than the core (dashed arrow). (F) Alcian blue staining of peripheral (left) and core (right) region of the construct (20×, scale bar = 100 µm).

Figure. 3. Schematics showing accessible surface area for nutrient/waste transport in gel-based constructs and novel strategies to enhance transport

(A–C) Nutrient transport paths under free swelling conditions; (A) Static culture, (B) static culture where constructs are regularly flipped (Flip over), (C) static culture where transport is improved by introduction of hollow fiber (HF) channels. (D–F) HF (and HF w/cotton threads)-mediated strategies to improve nutrient transport; (D) No channel, (E) Hollow fiber (HF), (F) HF w/ cotton threads. Schematics of paths available for nutrient/waste transport by HF or HF/ cotton threads. (pink (solid) arrow = nutrient “in”, blue (dashed) arrow = waste “out”).

Figure. 4. Gross appearance of tri-layered constructs with hollow fiber after 8 weeks of culture

The HF channel remained open during the culture period (A), and there was little to no interaction between the HF and the surrounding hydrogel (B) (scale bar = 1 mm).

Figure. 5. Maturation of tri-layered constructs with HFs

Groups are indicated as “No Channel” (left column), “Hollow Fiber” (HF; middle) and “HF w/threads” (right), respectively. (A) Gross appearance of tri-layered constructs with 8 weeks of culture (markers = 1 mm). (B) Cross-sectional view (markers = 1 mm). (C) CellTracker-labeled zonal chondrocytes and MSCs co-cultured in a tri-layered construct (2.5×, scale bar = 1 mm). Middle and bottom images are zoomed view of the interfaces of tri-layered constructs. (D) Calcein-AM staining of constructs at 8 weeks of culture (2.5×, scale bar = 1 mm).

Figure. 6. Histological analysis of tri-layered construct with long-term culture

(A) Alcian blue staining and immunohistochemistry of (B) type II collagen and (C) type I collagen of constructs at 16 weeks of culture (2.5× and 10× (inset), scale bar = 1mm (inset = 100 µm)). HF indicates original position of hollow fiber.

Figure. 7. Depth-dependent properties of tri-layered construct with long-term culture

(A–C) Bulk properties of tri-layered constructs: (A) E_Y (kPa; the baseline bulk E_Y of 1% MeHA is ~ 5 kPa.) (B) GAG (%WW), (C) Collagen (%WW) (Dashed gray line = native cartilage; Lighter bars = 8 weeks, Darker bars = 16 weeks). (D–E) Validation of a local compression device: (D) MSC-laden bi-layered HA constructs (1 and 5% MeHA) stained with Hoechst were subjected to compression (0% ~ 20% strain applied) and nuclei were tracked to compute local properties. Yellow arrows and circles indicate nuclei traced from reference image (0% strain) on day 0. (E) Local strain (ε_xx, Day 0). (F) Local E_Y (kPa) for tri-layered constructs at 16 weeks (No channel = blue, HF = red, HF w/cotton threads = green, Native cartilage = black, n=3/group). (p ≤ 0.05; * Native cartilage vs. Tri-layered constructs in the middle layer; † Bottom layer (#8) vs. Middle and Top layer).